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Free Flaps as the Vascularized Foundation for Hepatic Tissue Engineering
Peter A. Than, MD, Christopher R. Davis, BSc MB ChB, Kristine Rustad, MD, Paul A. Mittermiller, MD, Michael W. Findlay, MBBS PhD, Wei Liu, MD PhD, Alexander J. Whittam, BS, Sacha ML Khong, PhD, Kun Ma, MD PhD, Marc L. Melcher, MD PhD, Geoffrey C. Gurtner, MD.
Stanford University School of Medicine, Stanford, CA, USA.

PURPOSE: Creating patient-specific replacement organs promises to address shortcomings in transplantation, including the need for immunosuppression and limited donor supply. A major roadblock to tissue engineering is the inability to synthesize an intact circulatory system de novo. Thus, tissues produced by the current paradigm of seeding cellular scaffolds are rendered critically ischemic by their reliance on diffusion and eventual vascular ingrowth from the implantation site. Here, we describe a novel approach to fabricate vascularized neo-livers by repurposing the body’s own expendable microcirculatory beds (EMBs). The use of intact fully patterned circulatory beds, which are used clinically in free tissue transfer, allows immediate perfusion of the neo-livers after vascular anastomosis.
METHODS: Adipose derived stem cells (ASCs) were exposed to hepatic differentiation media. Hepatic differentiation was assessed by morphology, immunofluorescence, and western blot. In auto-assembly experiments ASCs, endothelial cells (ECs), and hepatocytes were co-cultured and assessed by bright field microscopy and immunofluorescence. Superficial inferior epigastric (SIE) flaps from male Wistar rats (n=3) were raised, detached at the vascular pedicle, and seeded with hepatocytes, ASCs, and ECs ex vivo. Following a two-hour dwell period, the constructs were re-implanted with vascular anastomoses to the femoral vessels. Subsequent neo-liver formation was assessed through a variety of modalities including histology and immunofluorescence.
RESULTS: ASCs exposed to differentiation media demonstrated a distinct morphology change, developing a hepatocyte-like appearance by day 21. Albumin and cytochrome P450 production was confirmed by immunofluorescence and western blot. Co-culture of hepatocytes, ASCs, and ECs recapitulates key developmental cues resulting in functional hepatic bud formation in vitro when assessed by morphology and immunofluorescence. EMBs seeded through intra-vascular perfusion of hepatocytes, ASCs, and ECs form vascularized neo-liver in vivo with sinusoidal architecture and function resembling native liver. These neo-livers express albumin, cytochrome P450, alpha-1-antitrypsin, and other hepatocyte-specific markers.
CONCLUSION: ASCs can be differentiated into hepatocyte-like cells, ensuring a renewable supply of autologous liver replacement cells. Co-seeding recapitulates the hepatic developmental environment and facilitates auto-assembly of functional liver tissue. Repurposing and seeding the body’s own microcirculatory beds with liver-forming cell combinations allows large, functional, vascularized neo-liver constructs to be implanted. This represents a major advancement in tissue engineering and paves the way for autologous organ replacement therapy.


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